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  • br In previous work we have shown that


    In previous work, we have shown that combined inhibition of EGFR, STAT3, and Src-YAP1 is more effective than single EGFR blockade or double combinations in culture and in vivo.9,11 In the current study, we investigated the effect of EGFR blockade on ALDH activity and the expression of CSCs and EMT markers. In a cohort of EGFR-mutationepositive NSCLC patients treated with EGFR TKIs, we explored the messenger RNA (mRNA) expression of HES1, ALDH1A1, ALDH1A3, and Bmi-1 as adverse effectors in EGFR-mutationepositive NSCLC patients.
    Patients and Methods
    Study Oversight and Sample Collection
    Clinical data were assessed in accordance with the protocol approved by the institutional review board of Germans Trias i Pujol Hospital, Badalona, and were deidentified for patient confidenti-ality. We studied remaining material from pretreatment tumors from a cohort of EGFR-mutationepositive NSCLC patients from hospitals in Spain and Colombia.9,11
    Cell Lines
    Human lung adenocarcinoma PC9 cells, harboring EGFR exon 19 deletion (E746-A750), were provided by Hoffmann-La Roche (Basel, Switzerland). Human lung adenocarcinoma H1975 cells, harboring both sensitizing L858R and resistant T790M mutation, were purchased from the American Type Culture Collection. Five osimertinib-resistant cell lines were generated by treating PC9 128-13-2 with increasing concentrations of osimertinib. Sequencing analyses 
    revealed that all 5 cell lines retained the EGFR exon 19 deletion. The half-maximal inhibitory concentration (IC50) for osimertinib of parental PC9 cells was in the nanomolar range compared to 3.1 to 3.7 mM in the resistant cell lines. All cell lines were maintained in RPMI 1640 medium supplemented with 1% penicillin/strepto-mycin/glutamine (Gibco; Thermo Fisher Scientific, Waltham, MA) and 10% fetal bovine serum (Gibco) in 5% CO2, 37 C cell culture incubator, and were routinely evaluated for mycoplasma contami-nation as previously described.9,11 Media and supplements were obtained from Life Technologies (Gaithersburg, MD).
    Chemical and Reagents
    Aldefluor Assay and Flow Cytometry
    The Aldefluor assay kit (STEMCELL Technologies, Vancouver, BC, Canada) was used to determine the profile of cells with high and low ALDH activity. The assay was performed according to the manufacturer’s instructions, with certain modifications. Briefly, 2 106 cells were suspended in Aldefluor assay buffer and divided into 2 groups. One group was pretreated for 10 minutes with the ALDH-specific inhibitor diethylaminobenzaldehyde (DEAB), and then both groups were incubated with ALDH enzyme substrate BODIPY-aminoacetaldehyde for 30 minutes at 37 C. Cells were then centri-fuged and resuspended in a fresh Aldefluor assay buffer to remove the unutilized substrate. The fluorescence intensity of stained cells was analyzed using a FACS canto II (BD Biosciences, San Jose, CA) flow cytometer. For the analysis, DEAB-treated sample was used as a negative control. The ALDH activity of a sample was determined to be high or low on the basis of fluorescence intensity above or below the threshold defined by the reaction with DEAB.
    Western Blot Test
    For Western blot assays, cells were cultured in cell culture flasks and left untreated or treated as indicated in each experiment. Cells were lysed in ice-cold radioimmunoprecipitation assay buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1
    Jordi Codony-Servat et al
    Figure 1 Effect of EGFR TKIs on STAT3 Activation in EGFR-MutationePositive Cells. PC9 Cells Were Cultured in Presence of (A) Gefitinib (40 nM) or (B) Osimertinib (0.17 mM), and H1975 Cells Were Cultured in Presence of (C) Osimertinib (0.17 mM) at Indicated Time Intervals; Western blot Analysis for various Signaling Molecules Was Performed. a-Tubulin Was Used as Housekeeping Protein. Independent Experiments Were Conducted at Least Twice
    Abbreviations: EGFR ¼ epidermal growth factor receptor; STAT3 ¼ signal transducer and activator of transcription 3; TKI ¼ tyrosine kinase inhibitor.
    mM b-glycerophosphate, 1 mM Na3VO4, 1 mg/mL leupeptin, 1 mM PMSF)]. After incubating for 20 minutes at 4 C, the samples were centrifuged, and the supernatant was kept at 80 C. Protein con-centration was determined by bicinchoninic acid protein assay. Equal amounts of protein from each cell lysate (30 mg per lane) were sub-jected to sodium dodecyl sulfateepolyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Milli-pore, New Bedford, MA). The membranes were blocked in Tris-buffered saline containing 5% fat-free dry milk and then probed with primary antibodies at 4 C overnight. After washing, the mem-brane was incubated with horseradish peroxidaseeconjugated